The aim of this application is to develop a new enabling technology for isolating circulating tumor cells (CTCs) from blood. During the last decade, there have been numerous publications describing the presence of CTCs in the blood of cancer patients. However, the methods have been insufficiently sensitive to serve as a cancer screening test to detect otherwise occult solid tumors. The only FDA- cleared CTCs test is approved solely for clinical staging, in identifying patients with many CTCs. Therefore, there is a need for a more sensitive CTCs sample preparation technology. We have developed the only antigen-dependent, negative selection technology for isolating CTCs from peripheral blood. Our technology is distinguished from existing methods by a uniquely high CTCs recovery rate, generally >90%, and a 5-6 log order enrichment. We attain these high rates through several sample preparation design innovations that minimize CTCs losses. For example, we do not introduce an external surface, such as a paramagnetic bead, that may interfere with downstream analysis. We have overcome the problem of limited EpCAM expression on CTCs associated with positive selection cell separation methods by designing ours as a negative selection technology. Our technology also is not dependent upon arbitrary physical distinctions between CTCs and blood cells, such as cellular density or sensitivity to chemical lysis. The technology is also unlike flow sorting, in that only basic laboratory instrumentation is required. Our method also avoids centrifugation, a procedure which, in our hands, consistently resulted in unacceptable CTCs losses. In this Phase I project, we will complete the development of our technology and test it with both model blood systems and patient samples. In Specific Aim 1 we will synthesize new reagent conjugates for removing leukocytes and erythrocytes from whole blood. We will also characterize the parameters of CTCs enrichment and evaluate entirely new methods of forming the reagent conjugates for scale-up to clinical trials (in Phase II). In Aim 2, we will characterize the efficacy of new cellular preservatives for maximizing CTCs viability during specimen transport. There is a dearth of information in the published literature on CTCs preservation in blood samples. In Aim 3, we will validate the assay with clinical samples from patients with prostate cancer and characterize whether it is substantially better than existing methods. If successful, this technology will establish a platform for detecting cancer at an earlier point in time, when surgical intervention may be curative. The best prospect for curative treatment of solid tumors is surgical excision, provided the tumor can be detected early, before it spreads. Unfortunately, all too many cancers are detected clinically only after they have spread. We propose to develop a novel technology for detecting extremely rare circulating tumor cells in blood, so as to facilitate early detection and curative surgical treatment. [unreadable] [unreadable] [unreadable]